Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute to cell migration and signaling by complex, integrated mechanisms. We are addressing the following specific questions: 1. What subcellular structures and signaling pathways are important for efficient cell migration in two-dimensional and three-dimensional environments? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce effective cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, live-cell phase-contrast and confocal time-lapse microscopy, and methods for direct measurement of intracellular hydrostatic pressure in a single migrating mammalian cell. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. We had recently discovered that for the newly characterized lobopodial mode of 3D cell migration, myosin II-dependent contractility generates high-pressure lobopodial protrusions at the front of various primary human cells migrating in a 3D matrix. In these cells, the nucleus physically compartmentalizes the cytoplasm into forward and rear compartments to maintain differences in hydrostatic pressure. Actomyosin contractility acting via vimentin and the nucleoskeleton-intermediate filament linker protein nesprin-3 pulls the nucleus forward and pressurizes the front of the cell for cell protrusion. In this mode of migration, the nucleus can act as a piston that physically compartmentalizes the cytoplasm and increases the hydrostatic pressure between the nucleus and the leading edge of a motile cell to drive lamellipodia-independent 3D lobopodial cell migration. This work has added an additional class of 3D cell migration to the previously characterized lamellipodial/mesenchymal and amoeboid forms of migration. Our ongoing work has been comparing normal and malignant cells for their use of this nuclear-piston mode of migration. Various types of fibrosarcoma cells appear to have lost this asymmetric pressure-based migration mechanism compared to their normal human fibroblast counterparts. We are examining the roles of integrin-based adhesions and proteases in this loss of nuclear piston function, as well as searching for an approach to restore this function to malignant cells. We previously demonstrated that myosin II could crosstalk to regulate levels of microtubule acetylation, though its biological significance was unclear. Our recent studies revealed that both fibroblasts and developing glands coordinate levels of cellular contractility and microtubule acetylation in a bi-directional, reciprocal fashion. Mechanistically, this balancing is achieved by competitive binding and dephosphorylation by myosin phosphatase of either myosin light chain or the key microtubule deacetylase HDAC6. Ongoing studies have been probing the tissue-specific mechanisms of this cytoskeletal regulatory process. Interestingly, the primary action of microtubule acetylation appears to be in the mesenchyme of embryonic salivary glands undergoing branching morphogenesis, which then regulates branching of the developing epithelium by signaling through secreted growth factors. Although cell-matrix adhesion and interactions have been studied extensively in cell culture on flat tissue culture substrates, much less is known about mechanisms of cell-matrix interaction and cytoskeletal functions in three-dimensional matrix micro-environments. We developed new methodologies for characterizing and quantifying the dynamics of individual cell-matrix adhesions on different forms of collagen fibrillar matrix. Stiff, bundled matrix increases cell-matrix adhesion stability and anchoring of cell protrusions. In contrast, adhesions slip back towards the cell in highly pliable matrices, and cell protrusions retract. The dynamics of 3D adhesions can be regulated locally by a combination of matrix rigidity, the strength/affinity of integrin-matrix attachment, and the extent of myosin II contractility; changing any of these parameters can alter the balance of matrix-to-adhesion coupling and adhesion lifespan. Unlike in regular tissue culture, inhibiting myosin II-mediated contractility stalls 3D migration in all matrix architectures. That is, regardless of the sizes of the matrix pores through which cells attempt to migrate in 3D collagen matrices, efficient migration requires myosin II contractility, suggesting that 3D migration requires some intrinsic cellular process dependent on contractility. Unexpectedly, we also found that force is not required for clustering of the activated conformation of integrins on 3D native collagen fibrils, which contrasts with findings using cells on flat 2D cell culture substrates and suggests the existence of an alternative mechanism regulating integrin activation and clustering in 3D microenvironments. This combined knowledge regarding the regulation of cell migration and phenotype in various microenvironments should provide novel approaches to understanding, preventing, or ameliorating migratory processes used by cells during abnormal embryonic development and particularly in cancer. An in-depth understanding of the precise manner in which cells move and interact with their matrix environment will also facilitate tissue engineering studies.